PROJECT SUMMARY/ ABSTRACT Strategies to prolong the durability of ART in low-and-middle income countries (LMICs) are important. With the new recommendation for dolutegravir to be used for all people living with HIV (PLHIV) globally, first-line ART now contains an agent with a high genetic barrier to resistance for the first time. Second-line ART still includes protease inhibitors (PIs) which have high genetic barriers to resistance like dolutegravir. First-line ART failure in the current era triggers enhanced adherence counseling, more frequent viral loads, and a switch to second- line ART if unsuccessful. Second-line ART failure in LMIC then requires expensive and difficult-to-access resistance testing and/or third-line ART and, therefore, unraveling the contribution of inadequate adherence to ART failure in LMICs, especially in the era of regimens with high genetic barriers to resistance, is critical. The AIDS Clinical Trials Group (ACTG) A5288 (MULTI-OCTAVE) study is an unprecedented prospective interventional study that evaluated strategies to treat PLHIV experiencing virologic failure on second-line regimens in LMIC. Self-reported adherence was not associated with virologic failure in the study, likely revealing the limitations of self-report. Importantly, A5288 chose to integrate an objective adherence metric into their study design by collecting hair samples for antiretroviral (ARV) levels, a methodology we pioneered at UCSF. However, A5288 requires independent funding to assay the hair samples (as sought in this R03) collected in the study to retrospectively assess the contribution of adherence to failure. Moreover, although performing hair levels is not practical in routine clinical settings, our group and others have recently developed and validated a practical, low-cost, real-time urine-based objective adherence metric, which measures tenofovir use over the short-term. We plan in this proposal to exploit a novel application of hair analysis by segmenting one half of each sample collected in A5288 into a proximal short segment (reflecting 1 week of exposure) and the other half into a longer segment to reflect longer-term (6 weeks) of exposure to examine both short-term and long-term metrics of adherence in relationship to virologic outcomes and resistance in LMIC. We will also examine the relationship between short-and long-term hair measures to determine if ?white coat? adherence occurred in this study. The use of objective adherence metrics in this large and well-characterized cohort of ART failure in LMIC will define the degree of adherence required to maintain virologic success on regimens with high genetic barriers to resistance (of particular import now that the new agent for first-line therapy globally is dolutegravir). Moreover, the novel design of this relatively low-cost proposal, leveraging data and samples already collected in an NIH-funded study, along with segmental hair analysis, will provide formative data on how to inform the use of practical short-term urine-based point-of-care adherence assays (which will be widely available within the year) to improve outcomes in resource-limited settings.